Potent Half-Sandwich Iridium(III) Anticancer Complexes ContainingC∧N‑Chelated and Pyridine LigandsZhe Liu,§ Isolda Romero-Canelon, Abraha Habtemariam, Guy J. Clarkson, and Peter J. Sadler*
Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, U.K.
*S Supporting Information
ABSTRACT: We report the synthesis and characterization of eight half-sandwich cyclopentadienyl IrIII pyridine complexes of the type [(η5-Cpxph)Ir(phpy)Z]PF6, in which Cpxph = C5Me4C6H5 (tetramethyl-(phenyl)cyclopentadienyl), phpy = 2-phenylpyridine as C∧N-chelatingligand, and Z = pyridine (py) or a pyridine derivative. Three X-ray crystalstructures have been determined. The monodentate py ligands blockedhydrolysis; however, antiproliferative studies showed that all the Ircompounds are highly active toward A2780, A549, and MCF-7 humancancer cells. In general the introduction of an electron-donating group(e.g., Me, NMe2) at specific positions on the pyridine ring resulted inincreased antiproliferative activity, whereas electron-withdrawing groups(e.g., COMe, COOMe, CONEt2) decreased anticancer activity. Complex5 displayed the highest anticancer activity, exhibiting submicromolarpotency toward a range of cancer cell lines in the National CancerInstitute NCI-60 screen, ca. 5 times more potent than the clinical platinum(II) drug cisplatin. DNA binding appears not to be themajor mechanism of action. Although complexes [(η5-Cpxph)Ir(phpy)(py)]+ (1) and [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]
+ (5) didnot cause cell apoptosis or cell cycle arrest after 24 h drug exposure in A2780 human ovarian cancer cells at IC50 concentrations,they increased the level of reactive oxygen species (ROS) dramatically and led to a loss of mitochondrial membrane potential(ΔΨm), which appears to contribute to the anticancer activity. This class of organometallic Ir complexes has unusual featuresworthy of further exploration in the design of novel anticancer drugs.
■ INTRODUCTION
The clinical use of platinum anticancer drugs has stimulated thesearch for other transition metal anticancer complexes withimproved features.1 In particular other platinum complexes2
and some group 8 metal complexes containing iron3 andruthenium4 centers show promising anticancer activity both invitro and in vivo.Very recently, possible biological applications of iridium
compounds have attracted attention.5 Half-sandwich organo-metallic IrIII compounds in particular display high versatilityand show promising anticancer activity.6 For example, Sheldricket al. have designed monoiridium and di-iridium polypyridylintercalators that target DNA in cancer cells.7 We have studieda series of half-sandwich IrIII anticancer agents of formula[(Cpx)Ir(L∧L′)Z]0/n+, where Cpx = Cp*, Cpxph (tetramethyl-(phenyl)cyclopentadienyl), or Cpxbiph (tetramethyl(biphenyl)-cyclopentadienyl), L∧L′ = bidentate ligand with nitrogen,oxygen, and/or carbon donor atoms, and Z = Cl, H2O, orpyridine (py).5a,6a We found that potent activity can beachieved by modification of ligands around the iridium centerand that small changes in structure can have a major effect onbiological activity. For example, antiproliferative activity asmeasured by IC50 values (concentration at which 50% of cellgrowth is inhibited) decreased dramatically from inactive (>100μM) to highly potent (submicromolar) when phenyl or
biphenyl was introduced in place of a methyl group on theCp* ring. We also reported that anticancer activity can beimproved significantly by replacement of neutral N∧N-chelatingligands with negatively charged C∧N-chelating ligands, leadingto increased cellular uptake and nucleobase binding.6c Themonodentate ligand Z (which in most of these IrIII half-sandwich compounds is Cl) is often readily substituted bywater in aqueous solution (hydrolysis), followed by interactionwith biological molecules. A relationship between hydrolysisand anticancer activity has been established for RuII arenecompounds, where readily hydrolyzed compounds are cytotoxicand those that do not hydrolyze are inactive or weakly activetoward cancer cells.8 For cyclopentadienyl Ir C∧N compounds,we found that decreasing hydrolysis by substitution of Cl bypyridine (py) does not result in loss of anticancer activity. Infact, the py complex is highly potent, ca. 10 times and 6 timesmore active than the clinically used platinum drug cisplatin(CDDP) and the chloride analogue, respectively.6a Theseresults encouraged us to explore in more detail the activity ofcomplexes containing py derivatives.In this study, the complexes contain Cpxph and C∧N-bound
2-phenylpyridine (phpy) as the cyclopentadienyl and chelating
Received: June 18, 2014Published: September 9, 2014
ligands, respectively, and various pyridine derivatives as themonodentate ligand Z. Thus, eight half-sandwich IrIII
compounds of the type [(η5-Cpxph)Ir(phpy)Z]PF6, where Z =pyridine or its derivatives, were synthesized and characterized.Their chemical behavior and antiproliferative activity towardcancer cells have been investigated.
nopyridine, methylnicotinate, N,N-diethylnicotinamide, 3-picoline, 4-picoline, 3-acetylpyridine, 9-ethylguanine, and 9-methyladenine werepurchased from Sigma-Aldrich. For the biological experiments, RPMI-1640 medium, fetal bovine serum, L-glutamine, penicillin/streptomycinmixture, trypsin/EDTA, and phosphate-buffered saline (PBS) werepurchased from PAA Laboratories GmbH. Cisplatin CDDP (≥99.9%),trichloroacetic acid (≥99%), sulforhodamine B (75%), sodiumphosphate monobasic monohydrate (≥99%), sodium phosphatedibasic heptahydrate (≥99%), acetic acid (≥99%), staurosporine,propidium iodide (>94%), and RNase A were obtained from Sigma-Aldrich. Complex [(η5-Cpxph)Ir(phpy)Cl] was prepared according toreported methods.6d
Syntheses. Compounds 1−8 were prepared by the same generalmethod: A solution of the chlorido complex [(η5-Cpxph)Ir(phpy)Cl]and AgNO3 (1 mol equiv) in MeOH and water (1:1, v/v) was heatedunder reflux in an N2 atmosphere for 3 h. The precipitate (AgCl) wasremoved by filtration through Celite, and pyridine derivative (10 molarequiv) was added to the filtrate. The reaction mixture was stirred atambient temperature for 12 h. NH4PF6 (10 mol equiv) was thenadded to the solution. The yellow precipitate was dissolved in acetone.The solution was evaporated slowly at ambient temperature, and thecrystalline product was collected by filtration, washed with diethylether, and recrystallized from methanol/diethyl ether.[(η5-Cpxph)Ir(phpy)(py)]PF6 (1). Yield: 76%.
+. Crystals suitable for X-ray diffraction wereobtained by slow evaporation of a methanol/acetone/water solution atambient temperature.
Methods and Instrumentation. X-ray Crystallography. Suitablecrystals of compounds 2, 5, and 8 were selected and mounted on aglass fiber with Fromblin oil on an Oxford Diffraction Gemini Xcaliburdiffractometer with a Ruby CCD area detector. The crystals were keptat 100(2) or 150(2) K during data collection. Using Olex2,9 thestructures of 2, 5, and 8 were solved with the XS10 structure solutionprogram using direct methods and refined with the XL10 refinementpackage using least squares minimization.
X-ray crystallographic data for compounds 2, 5, and 8 have beendeposited in the Cambridge Crystallographic Data Centre under theaccession numbers CCDC 1007223, 1007225, and 1007224,respectively.
NMR Spectroscopy. 1H NMR spectra were acquired in 5 mm NMRtubes at 298 or 310 K on either a Bruker DPX 400 (1H = 400.03MHz) or an AVA 600 (1H = 600.13 MHz) spectrometer. 1H NMRchemical shifts were internally referenced to CHD2OD (3.33 ppm) formethanol-d4 or CHCl3 (7.26 ppm) for chloroform-d1. MeOD-d4 wasused to aid solubility. All data processing was carried out usingMestReC or TOPSPIN version 2.0 (Bruker U.K. Ltd.).
Mass Spectrometry. Electrospray ionization mass spectra (ESI-MS)were obtained by preparing the samples in 50% CH3CN and 50% H2O(v/v) or using NMR samples for infusion into the mass spectrometer(Bruker Esquire 2000). The mass spectra were recorded with a scanrange of m/z 400−1000 for positive ions.
Elemental Analysis. CHN elemental analyses were carried out on aCE-440 elemental analyzer by Warwick Analytical (UK) Ltd.pH Measurement. pH or pH* values (pH meter reading without
correction for effect of deuterium on glass electrode) of NMR samplesin H2O or D2O were measured at ca. 298 K directly in the NMR tube,before and after recording NMR spectra, using a Corning 240 pHmeter equipped with a micro combination electrode calibrated withAldrich buffer solutions of pH 4, 7, and 10.Inductively Coupled Plasma Mass Spectrometry (ICP-MS). All
ICP-MS analyses were carried out on an Agilent Technologies 7500series ICP-MS instrument. The water used for ICP-MS analysis wasdoubly deionized (DDW) using a Millipore Milli-Q water purificationsystem and a USF Elga UHQ water deionizer. The iridium Specpureplasma standard (Alfa Aesar, 1000 ppm in 10% HCl) was diluted with5% HNO3 DDW to prepare freshly calibrants at concentrations of 50000, 10 000, 5000, 1000, 500, 200, 50, 10, and 5 ppt. The ICP-MSinstrument was set to detect 193Ir with typical detection limits of ca. 2ppt using no gas mode.Hydrolysis. Solutions of complexes 1−8 with final concentrations of
150 μM in 10% MeOD-d4/90% D2O (v/v) were prepared bydissolution of the complex in MeOD-d4 followed by rapid dilutionwith D2O.
1H NMR spectra were recorded after various time intervalsat 310 K.Interactions with Nucleobases. The reaction of complexes 1−8 (1
mM) with nucleobases 9-EtG or 9-MeA typically involved addition of1 mol equiv of nucleobase to an equilibrium solution of complexes 1−8 in 20% MeOD-d4/80% D2O (v/v). 1H NMR spectra of thesesolutions were recorded at 310 K after various time intervals.NCI-60 Screening. Compounds 2 and 5 were evaluated by the
National Cancer Institute Developmental Therapeutics Program(NCI/DTP, USA) for in vitro cytotoxicity toward ca. 60 humancancer cell lines. The cells were treated with iridium compounds for 48h at five concentrations ranging from 0.01 to 100 μM. Everycompound was tested twice, and data are the average of the twoexperiments. Data for cisplatin and oxaliplatin are from NCI/DTPscreening performed in October 2009 and 2010, respectively. Theprotocol for the determination of cytotoxicity toward the 60-cell-linepanel can be found at http://dtp.nci.nih.gov/branches/btb/ivclsp.html. The DTP homepage can be accessed at http://dtp.cancer.gov/.Cell Culture. A2780 ovarian carcinoma, A549 lung and MCF7
breast human adenocarcinoma cells were obtained from the EuropeanCollection of Cell Cultures (ECACC) and were grown in RoswellPark Memorial Institute medium (RPMI-1640) or Dubelco's ModifiedEagle Medium (DMEM). All media were supplemented with 10%(v/v) fetal calf serum, 1%(v/v) 2 mM glutamine, and 1% (v/v, 10k units/mL) penicillin/streptomycin. All cells were grown as adherentmonolayers at 310 K in a 5% CO2 humidified atmosphere andpassaged regularly at ca. 80% confluence.In Vitro Growth Inhibition Assay. Briefly, 5000 cells were seeded
per well in 96-well plates. The cells were preincubated in drug-freemedia at 310 K for 48 h before adding different concentrations of thecompounds to be tested. In order to prepare the stock solution of thedrug, the solid complex was dissolved first in 5% DMSO and thendiluted in a 50:50 v/v mixture of RPMI-1640/saline. This stock wasfurther diluted using cell culture medium until working concentrationswere achieved. The drug exposure period was 24 h. After this,supernatants were removed by suction, and each well was washed withPBS. A further 72 h was allowed for the cells to recover in drug-freemedium at 310 K. The SRB assay11 was used to determine cellviability. Absorbance measurements of the solubilized dye (on aBioRad iMark microplate reader using a 470 nm filter) allowed thedetermination of viable treated cells compared to untreated controls.IC50 values (concentration of drug resulting in a 50% cell growthinhibition) were determined as duplicates of triplicates in twoindependent sets of experiments, and their standard deviations werecalculated.Metal Accumulation in Cancer Cells. Iridium accumulation studies
for complexes 1 and 5 were conducted on A2780 ovarian cells. Briefly,1.5 × 106 cells were seeded on a six-well plate. After 24 h ofpreincubation time in drug-free medium at 310 K, the complexes were
added to give final concentrations equal to IC50, and a further 24 h ofdrug exposure was allowed. After this time, excess drugs were removedby suction, and cells were washed with PBS and then treated withtrypsin-EDTA. A suspension of single cells was counted, and cellpellets were collected. Each pellet was digested overnight inconcentrated nitric acid (73%) at 353 K; the resulting solutionswere diluted with double-distilled water to a final concentration of 5%HNO3, and the amount of Ir taken up by the cells was determined byICP-MS. These experiments did not include any cell recovery time indrug-free media; they were carried out in triplicate, and the standarddeviations were calculated.
Cell Cycle Analysis. A2780 cells at 1.5 × 106 per well were seeded ina six-well plate. Cells were preincubated in drug-free media at 310 Kfor 24 h, after which drugs were added at equipotent concentrationsequal to IC50. After 24 h of drug exposure, supernatants were removedby suction and cells were washed with PBS. Finally, cells wereharvested using trypsin-EDTA and fixed for 24 h using cold 70%ethanol. DNA staining was achieved by resuspending the cell pellets inPBS containing propidium iodide (PI) and RNAse. Cell pellets werewashed and resuspended in PBS before being analyzed in a BectonDickinson FACScan flow cytometer using excitation of DNA-boundPI at 536 nm, with emission at 617 nm. Data were processed usingFlowjo software.
Induction of Apoptosis. Flow cytometry analysis of apoptoticpopulations of A2780 cells caused by exposure to complexes 1 and 5was carried out using the annexin V-FITC apoptosis detection kit(Sigma-Aldrich) according to the supplier’s instructions. Briefly, 1.5 ×106 A2780 cells per well were seeded in a six-well plate. Cells werepreincubated in drug-free media at 310 K for 24 h, after which drugswere added at equipotent concentrations equal to IC50. After 24 h ofdrug exposure, supernatants were removed by suction, and cells werewashed with PBS. Finally, cells were harvested using trypsin-EDTA.Sample staining was achieved by resuspending the cell pellets in buffercontaining annexin V-FITC and PI. For positive-apoptosis controls,A2780 cells were exposed to staurosporine (1 μg/mL) for 2 h. Cellsfor apoptosis studies were used with no previous fixing procedure as toavoid nonspecific binding of the annexin V-FITC conjugate.
ROS Determination. Flow cytometry analysis of ROS/superoxidegeneration in A2780 cells caused by exposure to complexes 1 and 5was carried out using the Total ROS/Superoxide detection kit (Enzo-Life Sciences) according to the supplier’s instructions. Briefly, 1.5 ×106 A2780 cells per well were seeded in a six-well plate. Cells werepreincubated in drug-free media at 310 K for 24 h in a 5% CO2humidified atmosphere, and then drugs were added to triplicates atconcentrations of IC50 and 2 × IC50. After 1 h of drug exposure,supernatants were removed by suction and cells were washed andharvested. Staining was achieved by resuspending the cell pellets inbuffer containing the orange/green fluorescent reagents. Cells wereanalyzed in a Becton Dickinson FACScan flow cytometer using FL1channel Ex/Em: 490/525 nm for the oxidative stress and FL2 channelEx/Em: 550/620 nm for superoxide detection. Data were processedusing Flowjo software. At all times, samples were kept under darkconditions to avoid light-induced ROS production.
Mitochondrial Membrane Assay. Analysis of the changes ofmitochondrial potential in A2780 cells after exposure to complexes 1and 5 was carried out using the Abcam, JC-10 mitochondrialmembrane potential assay kit according to the manufacturer’sinstructions. Briefly, 1.5 × 106 cells were seeded in six-well platesleft to incubate for 24 h in drug-free medium at 310 K in a humidifiedatmosphere. Drug solutions, at equipotent concentrations equal toIC50 and 2 × IC50, were added in triplicate, and the cells were left toincubate for a further 24 h under similar conditions. Supernatants wereremoved by suction, and each well was washed with PBS beforedetaching the cells using trypsin-EDTA. Staining of the samples wasdone in flow cytometry tubes protected from light, incubating for 30min at ambient temperature. Samples were immediately analyzed on aBeckton Dickinson FACScan, reading the reduction of fluorescence inthe FL2 channel. For positive controls, A2780 cells were exposed tocarbonyl cyanide 3-chlorophenylhydrazone, CCCP (5 μM), for 15min. Data were processed using Flowjo software.
■ RESULTSNovel Ir compounds 1−8 were synthesized in moderate yieldsfrom the chlorido analogue [(η5-Cpxph)Ir(phpy)Cl]6d bysubstitution of chloride by pyridine or its derivatives in thepresence of silver nitrate, Scheme 1. All the synthesized
complexes were isolated as PF6− salts and were fully
characterized by 1H NMR spectroscopy, CHN elementalanalysis, and ESI-MS. The complexes studied in this work areshown in Scheme 1.X-ray Crystal Structures. The X-ray crystal structures of
[(η5-Cpxph)Ir(phpy)(4-Me-py)]PF6 (2), [(η5-Cpxph)Ir(phpy)-(4-NMe2-py)]PF6 (5), and [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8) were determined. The complexes adopt theexpected half-sandwich pseudo-octahedral “three-leg piano-stool” geometry with the Ir bound to a η5-cyclopentadienylligand occupying three coordination sites, the nitrogen atom ofthe py derivative (2.099−2.118 Å), and a 2-phenylpyridineC∧N-chelating ligand. Their structures are shown in Figure 1.Crystallographic data are shown in Table S1, and selected bondlengths and angles are listed in Table 1.The crystal structures reported here are the second examples
of crystal structures containing the Cpxph ligand.6b The phenyl
ring of Cpxph is twisted by about 45° relative to thecyclopentadienyl ring in 2 and 5 and 83° in 8. The Ir−cyclopentadienyl (centroid) bond distances for compounds 2,5, and 8 ranged from 1.825 to 1.832 Å, longer than that of [(η5-Cpxph)Ir(bpy)Cl]PF6 (bpy = 2,2′-bipyridine)6b (1.789 Å),probably due to the negatively charged phpy ligand. Thechange in monodentate ligands in 2, 5, and 8 does not give riseto much difference in bond lengths between Ir and coordinatedatoms; however, a smaller N−Ir−N angle of 80.87(9)° for 8 isobserved compared to 88.27(9)° and 87.01(6)° for 2 and 5,respectively. There is weak π−π intermolecular ring stackingbetween the neighboring phenylpyridine rings in the unit cell ofcompound 2, Figure S1. The two interacting π systems areparallel, with a centroid−centroid distance of 4.291 Å.
Hydrolysis. The hydrolysis of complexes 1−8 (150 μM) in10% MeOD-d4/90% D2O (v/v) was studied by 1H NMRspectroscopy at 310 K. The presence of methanol ensuredsufficient solubility of the complex. The 1H NMR spectrashowed no obvious change over 24 h, indicating that these Ircompounds remained stable under these conditions.
Antiproliferative Activity. The activity of complexes 1−8toward A2780 human ovarian, A549 lung, and MCF-7 breastcancer cells was investigated, Table S2 and Figure 2. The IC50values (concentration at which 50% of the cell growth isinhibited) for all IrIII complexes are comparable to or lower
Scheme 1. Synthesis of Ir Compounds Studied in This Work
Figure 1. X-ray crystal structures for (A) [(η5-Cpxph)Ir(phpy)(4-Me-py)]PF6 (2), (B) [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]PF6 (5), and (C) [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8), with thermal ellipsoids drawn at 50% probability. The hydrogen atoms and counterions have been omittedfor clarity.
Table 1. Selected Bond Lengths (Å) and Angles (deg) for[(η5-Cpxph)Ir(phpy)(4-Me-py)]PF6 (2), [(η
5-Cpxph)Ir(phpy)(4-NMe2-py)]PF6 (5), and [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8)
2 5 8
Ir−C 2.170(3) 2.1676(19) 2.163(3)
(cyclopentadienyl) 2.173(2) 2.1727(18) 2.168(3)
2.202(3) 2.1766(19) 2.168(3)
2.223(3) 2.2316(18) 2.245(3)
2.236(3) 2.2442(18) 2.263(3)
Ir−C(centroid) 1.827 1.825 1.832
Ir−C(phpy) 2.065(2) 2.0505(17) 2.053(3)
Ir−N*a 2.073(2) 2.0811(16) 2.093(3)
Ir−N#b 2.106(2) 2.0994(15) 2.118(2)
C−Ir−N* 78.34(9) 78.40(6) 78.15(12)
C−Ir−N# 85.94(8) 84.66(6) 86.67(10)
N*−Ir− N# 88.27(9) 87.01(6) 80.87(9)aN* is the nitrogen atom in the 2-phenylpyridine chelating ligand. bN#
than that of cisplatin, suggesting that all these compounds arehighly active. Complex 5, [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]
+,containing 4-dimethylaminopyridine, displayed the highestanticancer activity, with an IC50 value of 0.20 ± 0.04 μMtoward MCF-7 cells, ca. 36 times more potent than cisplatin.Complex 8, containing N,N-diethylnicotinamide, showed thelowest anticancer activity toward all three cancer cell lines.With regard to the effects of substitutions on the pyridine
ring on anticancer activity, overall, complexes containingelectron-withdrawing groups on the pyridine ring show lessactivity compared to those complexes with an electron-donating group.The antiproliferative activity of compounds 2 and 5 was
further evaluated in the National Cancer Institute NCI-60human cancer cell screen, consisting of nine tumor subtypes.12
Three end points were determined: GI50 (the concentrationthat causes 50% cell growth inhibition), TGI (the concen-tration that causes 100% cell growth inhibition), and LC50 (theconcentration that decreases the original cell number by 50%).The GI50 mean graph for 2 and 5 is shown in Figure 3. Themidpoint (log10 GI50) of 2 and 5 is −6.14 (GI50 = 724 nM) and−6.46 (GI50 = 347 nM), respectively. Bars extending to the leftin the mean graph represent higher activity than the mean of alltested cell lines. Bars extending to the right correspondinglyimply activity less than the mean. Complex 5 shows highpotency in a wide range of cancer cell lines (Figure 3), withparticular selectivity toward MDA-MB-468 (breast), A498(renal), and COLO-205 (colon), with GI50 values of <170
nM. Notably, complex 2 displayed potency toward the A498(renal) cell line with a GI50 of 19 nM. Complex 5 showed goodselectivity toward leukemia, CNS cancer, colon cancer, andbreast cancer. In comparison with cisplatin (CDDP), Ircomplexes displayed higher activity against NCI-60 cancercell lines, especially Ir complex 5, which is 4−5 times moreactive than cisplatin, Figure 4.
Interactions with Nucleobases. Reactions of complexes1−8 with nucleobase derivatives 9-ethylguanine (9-EtG) and 9-methyladenine (9-MeA) were investigated. Solutions of 1−8(ca. 1 mM) and 1 molar equiv of 9-EtG or 9-MeA in 20%MeOD-d4/80% D2O (v/v) were prepared, and 1H NMRspectra were recorded at different time intervals at 310 K.No reaction with 9-MeA was observed for all complexes, as
addition of nucleobase model to a solution of 1−8 resulted inno additional 1H NMR peaks over 24 h. In contrast, all thecomplexes reacted with 9-EtG. For example, in the 1H NMRspectrum of a solution containing 8 and 1 molar equiv of 9-EtG, one set of new peaks assignable to the 9-EtG adduct 8Gappeared, showing that 32% of 8 had reacted after 24 h (Figure5). A significant change in chemical shift of the CHN (phpyligand) proton of complex 8 from 8.88 to 9.29 ppm for 8G wasobserved. A new 9-EtG H8 peak appeared at 7.43 ppm(singlet), shifted by 0.39 ppm to high field relative to that offree 9-EtG. The ESI-MS of an equilibrium solution contained amajor peak at m/z 723.2, confirming the formation of the 9-EtG adduct 8G, [(η5-C5Me4C6H5)Ir(phpy)(9-EtG)]
+ (calcdm/z 722.9). The percentages of nucleobase adducts formed byall the complexes after 24 h reaction, based on 1H NMR peakintegrals, are shown in Table S3 and Figure 6.
Cellular Ir Accumulation. Complex 5, which displayed thehighest anticancer activity, and complex 1, containing anonsubstituted py ligand, were selected for further studies.First we investigated the cellular accumulation of Ir fromcomplexes 1 and 5 in A2780 ovarian cancer cells. After 24 h ofdrug exposure at equipotent concentrations corresponding toIC50 values, 3.5 times more Ir, as determined by ICP-MS, fromthe pyridine complex 1 was accumulated in the cells comparedto the py-NMe2 analogue 5 (7.8 ± 0.5 ng of Ir vs 2.2 ± 0.3 ngof Ir per 106 cells).
Apoptosis Assay. In order to investigate whether thereduction in cell viability observed by the SRB assay is based onapoptosis, A2780 cells were treated with complexes 1 and 5 atequipotent concentrations of IC50 for 24 h, then stained withannexin V/propidium iodide and analyzed by flow cytometry.This allowed determination of cell populations as viable(unstained, only self-fluorescence), early apoptosis (stained byannexin V only, green fluorescence), late apoptosis (stained byannexin V and PI, green and red fluorescence), and nonviable(stained by PI only, red fluorescence). Dot plots (Figure 7 andTable S4) showed that around 95% of A2780 cells remained inthe viable phase after 24 h of exposure to 1 and 5, indicating noobvious induction of apoptosis at equipotent concentrations ofIC50.
Cell Cycle Studies. Next we performed cell cycle arrestanalysis for complexes 1 and 5 toward A2780 cells by flowcytometry to determine whether the induced cell growthinhibition was the result of cell cycle arrest. In comparison tothe control population, the cell cycle data (Figure 8 and TableS5) clearly show no significant population change, indicatingthat Ir compounds 1 and 5 did not cause cell cycle arrest atequipotent concentrations of IC50.
Figure 2. Inhibition of growth of (A) A2780 human ovarian cancer;(B) A549 lung cancer; and (C) MCF-7 breast cancer cells bycompounds 1−8 and comparison with cisplatin (CDDP).
Induction of ROS in A2780 Cancer Cells. Wedetermined the level of reactive oxygen species (ROS) inA2780 human ovarian cancer cells induced by complexes 1 and5 at concentrations of IC50 and 2 × IC50 by flow cytometry
fluorescence analysis (Figure 9 and Table S6). This allowed thedetermination of the total level of oxidative stress (combined
Figure 3. NCI-60 GI50 mean graphs for complexes 2 (right) and 5 (left). The midpoint (log10 GI50) is −6.14 (2) and −6.46 (5). Bars to the right ofthe mean indicate lower activity relative to the mean, and those to the left, higher activity.
Figure 4. GI50, TGI, and LC50 values (μM) of 2, 5, and CDDP in theNCI-60 screen.
Figure 5. Low-field region of the 1H NMR spectra for reaction of [(η5-Cpxph)Ir(phpy)(3-CONEt2-py)]PF6 (8) with 9-EtG: (A) 10 min afteraddition of 1 mol equiv of 9-EtG to an equilibrium solution of complex8 (1.0 mM) in 20% MeOD-d4/80% D2O (v/v) at 310 K; (B) after 24h reaction. Peak assignments: (red squares) 8; (blue squares) guanineadduct 8G. After 24 h, 32% of 8 had reacted.
levels of H2O2, peroxy and hydroxyl radicals, peroxynitrite, andNO), while also monitoring superoxide production. After only1 h of drug exposure, we observed a dramatic increase in bothtotal ROS levels and superoxide levels in cells treated withcomplexes 1 and 5 compared to untreated cells. ROS weredetected in more than 97% of A2780 cells. A concentrationdependence of ROS induction was observed for both Ircomplexes: the population of cells that shows high fluorescencein both FL-1 and FL-2 channels (indicating high total oxidativestress as well as high superoxide levels) increased from 48 ± 2%at IC50 to 64 ± 3 at 2 × IC50 for complex 1 and increased from40 ± 3% at IC50 to 47 ± 2 at 2 × IC50 for complex 5 (Figure 9and Table S6).
Polarization of the Membrane Potential. Analysis of thechanges of mitochondrial membrane potential (ΔΨm) inA2780 cells after exposure to complexes 1 and 5 was carried outby observing the fluorescence of JC-10, a cationic lipophilic dye,using flow cytometry. JC-10 aggregates inside mitochondria andemits red fluorescence; however, upon membrane polarization,JC-10 is disaggregated, reducing the red emission. The level ofmembrane polarization after cells were exposed to complexes 1and 5 at concentrations of IC50 and 2 × IC50 for 24 h is shown
Figure 6. Bar chart showing the extent of binding of complexes 1−8(ca. 1 mM in 20% MeOD-d4/80% D2O) to the nucleobase 9-EtG over24 h at 310 K.
Figure 7. Apoptosis analysis of A2780 human ovarian cells after 24 hof exposure to complexes 1 and 5 at 310 K determined by flowcytometry using annexin V-FITC vs PI staining. (A) FL1 vs FL2histogram for cells exposed to complexes 1 and 5 at equipotentconcentrations of IC50. (B) Populations for cells treated by 1 and 5. p-Values were calculated after a t test against the negative control data,*p < 0.05, **p < 0.01, ap > 0.05.
Figure 8. Cell cycle analysis of A2780 human ovarian cancer cells after24 h of exposure to complexes 1 and 5 at 310 K. Concentrations usedwere equipotent at IC50. Cell staining for flow cytometry was carriedout using PI/RNase. (A) FL2 histogram for negative control (cellsuntreated) and complexes 1 and 5. (B) Cell populations in each cellcycle phase for control and complexes 1 and 5. p-Values werecalculated after a t test against the negative control data, *p < 0.05, **p< 0.01, ap > 0.05.
Figure 9. ROS induction in A2780 cancer cells treated with complexes1 and 5. FL1 channel detects total oxidative stress, and FL2 channeldetects superoxide production. (A) Comparison between the fourdifferent populations caused by IC50 and 2 × IC50 of 1. (B)Comparison between the four different populations caused by IC50and 2 × IC50 of 5. p-Values were calculated after a t test against thenegative control data, *p < 0.05, **p < 0.01, ap > 0.05.
in Figure 10 and Table S7. Both Ir complexes have significanteffects on cell membrane polarization; around 70% of cells lost
ΔΨm. The impairment induced by 1 and 5 (which is reflectedin ΔΨm) is clearly concentration-dependent (Figure 10).
■ DISCUSSIONIrIII complexes are often considered to be relatively inert, acommon characteristic of low-spin d6 metal ions and especiallythird-row transition metals.13 Compared to platinum- orruthenium-based anticancer agents, iridium anticancer com-plexes are still in their infancy.With regard to half-sandwich IrIII complexes [(Cpx)Ir(L∧L′)-
Z]0/n+, we found that both the cyclopentadienyl Cpxph orCpxbiph ligand and chelating ligand C∧N− can dramaticallyinfluence anticancer activity.6b−d In addition, we have shownthat complexes containing pyridine as the monodentate ligandexhibit 6 times higher anticancer activity compared to thechlorido analogue.6a Therefore, we have investigated a series ofIrIII complexes of type [(Cpxph)Ir(phpy)Z]+ containing phenyl-substituted Cp*, C∧N-bound 2-phenylpyridine, and pyridine orits derivatives (Scheme 1) in this work. Novel compounds 1−8have been synthesized and are reported for the first time.Encouragingly, all eight compounds exhibit high potency
against human ovarian A2780 cancer, A549 lung cancer, andMCF-7 breast cancer cells, at least comparable with cisplatin,Figure 2. In general an electron-donating substituent on the pyring confers higher activity in comparison with electron-withdrawing groups. This may arise from strengthening the Ir−N(py) bond, thus reducing side reactions on the way to targetsites. In addition, lipophilicity might as well influence the
potency of these complexes.14 Complex 5, [(η5-Cpxph)Ir(phpy)-(4-NMe2-py)]
+, showed the highest anticancer activity, ca. 3−9times more active than unmodified py complex 1. In addition,complex 5 shows submicromolar activity toward a wide rangeof cancer cell lines in the NCI-60 screen, with selectivity forleukemia, CNS cancer, colon cancer, and breast cancer celllines, being 4−5 times more potent than CDDP (Figures 3 and4). Cpxph py complex 1 displayed ca. 8 times less anticancerpotency than the Cpxbiph analogue, which is consistent with thegeneral finding we reported previously that the anticancerefficiency increases with phenyl substitution on the Cp*ring.6a,b,d
Hydrolysis often presents an activation step for transitionmetal anticancer complexes.15 However, no significanthydrolysis was observed for complexes 1−8 in aqueoussolution. DNA is usually a potential target for transitionmetal anticancer drugs.16 Although 1−8 are inert to hydrolysis,they can react with nucleobase 9-EtG to various extents from11% to 50% (Figure 6), depending on the electronic effect ofthe substituent on the py ring. Electron-withdrawing groups(such as acetyl and ester groups) facilitate ligand substitution ofthe py derivative by 9-EtG, whereas electron donor groups(such as methyl and dimethylamino groups) hamper formationof the Ir-EtG adduct. No reaction of 1−8 with 9-MeA wasobserved, consistent with our previous studies that guaninebinds stronger to IrIII than adenine.6a,b,d The extent ofnucleobase binding does not correlate with antiproliferativeactivity. Compared to complexes 1−5, complexes 6−8 bind to9-EtG to a greater extent; however, they show lower activitytoward cancer cells. Therefore, although DNA is a potentialtarget for these iridium compounds, DNA binding may not bethe major mechanism of action.Apoptosis is a process of cell death in a programmed fashion.
A large number of transition metal-based anticancer agentshave been reported to inhibit cell growth by activation ofapoptosis.17 Induction of apoptosis is usually dependent on theconcentration of administered compounds17f,18 and onexposure time.19 No apoptosis was observed when A2780cells were exposed to complexes 1 and 5 at their IC50concentrations for 24 h. Also IC50 concentrations of complexes1 and 5 did not cause significant cell cycle arrest after 24 h ofdrug exposure. Lack of accumulation of cells in the sub-G1phase in cell cycle experiments is consistent with the absence ofapoptotic cell death.20
Reactive oxygen species play important roles in regulatingcell proliferation, death, and signaling. They can also playsignificant roles in the mechanism of action of anticanceragents.21 In fact, dinuclear Cp*Ir(III) complexes containingbridging dipyridyl ligands have been reported to generate ROSand induce apoptosis in Jurkat leukemia cells.17f We suggestedpreviously that the antiproliferative mechanism for the iridiumpyridine complex in this series is related to ROS generation.6a
Consequently, we also determined the ROS levels in A2780ovarian cancer cells induced by 1 and 5. Both complexesincreased ROS levels significantly even at IC50 concentrationafter 1 h drug exposure (Figure 9), which led to the majority ofcancer cells (>97%) being affected by generation of ROS.These increases in ROS levels may provide a basis for killingcancer cells.Mitochondria are involved in a number of important tasks in
living cells, such as energy production and generation of ROS.Mitochondrial dysfunction can participate in the induction ofcell death and was assessed by measuring changes in the
Figure 10. Changes in mitochondrial membrane potential of A2780human ovarian cancer cells induced by complexes 1 and 5. (A) Flowcytometry histograms of the changes induced by the complexes atconcentrations of IC50 and 2 × IC50. (B) Populations of cells thatexhibit a reduction in the FL2 fluorescence, indicative of changes inthe mitochondrial membrane potential. p-Values were calculated aftera t test against the negative control data, **p < 0.01.
mitochondrial membrane potential. Intriguingly, both com-plexes 1 and 5 (IC50 concentration) induced significant changesin mitochondrial membrane potential (Figure 10); more than70% of A2780 cells lose ΔΨm after exposure to Ir compoundsfor 24 h. This may contribute to the anticancer activities ofthese Ir compounds.
■ CONCLUSIONSIn this work, we have prepared eight new organometallic IrIII
cyclopentadienyl complexes [(η5-Cpxph)Ir(phpy)Z]PF6 toexplore the effect of a monodentate pyridine-based ligand ontheir chemical and anticancer activity. The X-ray crystalstructures of [(η5-Cpxph)Ir(phpy)(4-Me-py)]PF6 (2), [(η5-Cpxph)Ir(phpy)(4-NMe2-py)]PF6 (5), and [(η5-Cpxph)Ir-(phpy)(3-CONEt2-py)]PF6 (8) were determined.All the complexes display high potency toward A2780, A549,
and MCF-7 human cancer cells, comparable to, and for somecomplexes even higher than, the clinical anticancer drugcisplatin. The anticancer activity can be fine-tuned by varyingthe pyridine-based ligand; the presence of an electron-donatinggroup confers higher anticancer activity. The most activecomplex, 5, contains a 4-dimethylamine substituent onpyridine. The results of the NCI 60 cancer cell line screeningshow that complex 5 is 4−5 times more potent than cisplatinand exhibits submicromolar activity in a wide range of cancercell lines, especially against leukemia, CNS cancer, coloncancer, and breast cancer. Nanomolar activity (GI50 19 nM)was obtained for complex 2 toward the renal A498 cancer cellline.No distinct hydrolysis was observed for this type of complex
in aqueous solution; however, all complexes display weaknucleobase binding to 9-ethylguanine, suggesting that DNAcould be a possible target, although other targets appear to bemore important. Additionally, no obvious apoptosis and cellcycle arrest were induced when A2780 cancer cells were treatedwith IC50 concentrations of complexes 1 and 5. However, theiridium complexes 1 and 5 induce a dramatic increase in thelevel of ROS in ovarian cancer cells within 1 h and causedmitochondrial dysfunction by loss of the mitochondrialmembrane potential. Our work suggests that this type ofiridium complex could be a promising candidate for furtherevaluation as chemotherapeutic agents for human cancers.
■ ASSOCIATED CONTENT*S Supporting InformationThis material is available free of charge via the Internet athttp://pubs.acs.org.
■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected] Address§Department of Chemistry, University of Basel, Spitalstrasse 51,4056 Basel, Switzerland.NotesThe authors declare no competing financial interest.
■ ACKNOWLEDGMENTSWe thank the ERC (Grant No. 247450), Science City (AWM/ERDF), and the EPSRC for support, the National CancerInstitute for 60 human tumor cell screening, and members ofEU COST Actions D39 and CM1105 for stimulating
discussions. We also thank Dr. Magdalena Mos and MissBushra Qamar for assistance with cell culture.
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